Supplementary Material
We previously described the construction of a ura3 deletion in C. parapsilosis (Ding
& Butler, 2007) and here we used a similar approach to generate a his1/ura3 strain.
HIS1 was disrupted using the SAT1-flipper cassette (Ding & Butler, 2007). Due to the
low efficiency of integration at HIS1, the two alleles were disrupted using different
constructs (pCD35 and pCD37). For plasmid pCD35, a 405-bp fragment from the
upstream region of HIS1 (including 354 bp of promoter sequence and 51 bp coding
sequence) was amplified using oligonucleotides BUT316 and BUT317, introducing
restriction sites KpnI and ApaI respectively, which was then cloned into plasmid
pCD8 (Ding & Butler, 2007), generating pCD33. A 423-bp fragment from the
downstream region of HIS1 was amplified using oligonucleotides BUT318 and
BUT319, and was cloned between restriction sites SacII and SacI on plasmid pCD33
to generate pCD35. The SAT1 cassette was isolated from plasmid pCD35 by digestion
with KpnI and SacI, and the fragment was introduced into ura3 deletion strain CDU1
to delete the first HIS1 allele. The SAT1 cassette was then recycled from CDUhis1,
generating the heterozygote knockout CDUhis11. For plasmid pCD37, a 439-bp
fragment from the upstream region of HIS1 (including 110 bp of promoter sequence
and 329 bp of coding sequence) was amplified from C. parapsilosis genomic DNA
using oligonucleotides HisKpn2 and HisApa2, and the fragment was cloned between
restriction sites KpnI and ApaI in plasmid pCD35 to generate plasmid pCD37. The
cassette was released from pCD37 by digestion with KpnI and SacI, and the purified
fragment was introduced into CDUhis11 to generate strain CDUH1. Recycling of the
cassette from CDUH1 generated CDUH3.
Verification of gene disruption by PCR and Southern blot analysis
Southern blots were used to confirm the his1 deletion. 20 g of genomic DNA
from the wildtype and HIS1 knockout strains (CLIB214, CDUhis1, CDUhis11,
CDUH1, and CDUH3) were digested with HincII. A probe was amplified from C.
parapsilosis genomic DNA using BUT318 and BUT319, which binds to a region of
HIS1 downstream from the integration site. Labeling and hybridizations were carried
out using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche).
The BCR1 knockout generated using URA3 and HIS1 was confirmed by PCR.
Oligonucleotides BUT261 binds upstream from BCR1 and URAR binds within the
URA3 cassette, generating a fragment of 1.25 kb. Oligonucleotides BUT261 and
HISR, which bind within HIS1, generate a fragment of 1.25 kb. Oligonucleotide
BUT261 and BUT262 (from within the BCR1 ORF) were used to confirm the deletion
of both alleles.
Figure S1. Deleting HIS1 and BCR1 in C. parapsilosis.
A. HIS1 was knocked out in a ura3 null strain of C. parapsilosis (CDU1 (Ding &
Butler, 2007)) using the SAT1-flipper cassette, to generate a ura3his1
strain (CDUH3).Two different constructs were used to disrupt the two HIS1
alleles, as described in Methods. Genomic DNA was isolated from five
strains, digested with HincII and hybridized with a probe from the
downstream region from HIS1. The wildtype allele generates a1.0 kb
fragment. Integration of the cassettes at HIS1 produces a 2.0 kb fragment.
Recycling of the first cassette generates a 3.7 kb fragment, and recycling of
the second cassette results a 4.0 kb fragment. Lane 1: C. parapsilosis
CLIB214; Lane 2: C. parapsilosis CDUhis1 (his1::SAT1-FLP/ HIS1); Lane
3: C. parapsilosis CDUhis11 (his1::FRT/ HIS1); Lane 4: C. parapsilosis
CDUH1 (his1::FRT/ his1::SAT1-FLP); and Lane 5: C. parapsilosis
CDUH3 (his1::FRT/ his1::FRT). All strains (apart from CLIB214) are also
carrying ura3 deletions.
B. BCR1 was disrupted by replacement with URA3 and HIS1 genes from C.
albicans, amplified by PCR and transformed into C. parapsilosis CDUH3 by
electroporation. Integration of the HIS3 gene was confirmed by PCR using
oligonuleotides But261 and HISR that generates a 1.26 kb fragment which is
absent in the wild-type strain. Integration of URA3 was confirmed using
oligonuleotides But261 and URAR, which generates a 1.25 kb fragment. The
deletion of BCR1 was confirmed using oligonucleotides But261 and But262,
which generate a 0.8 kb fragment from the wildtype allele. Lanes 1,4,7:
But261/HISR; Lanes 2,5,8: But261/URAR; Lanes 3,6, 9: But261/But262. WT
= CDUH3, CDUHBhis (bcr1::HIS1/BCR1), CDUHB6 (bcr1::HIS1/
bcr1::URA3).
Ding, C. & G. Butler, (2007) Development of a gene knockout system in Candida
parapsilosis reveals a conserved role for BCR1 in biofilm formation. Eukaryot
Cell 6: 1310-1319.